Homozygous 6-bp deletion of IGFALS in a prepubertal boy with short stature

Biallelic IGFALS variants lead to acid‒labile subunit (ALS) deficiency characterized by growth hormone resistance with or without delayed puberty. Here, we report a prepubertal boy with a homozygous 2-amino acid deletion within the fourth N-glycosylation motif (c.1103_1108del, p.N368_S370delinsT) associated with parental consanguinity. He showed short stature consistent with ALS deficiency. This case expands the mutation spectrum of IGFALS to include the elimination of only one N-glycosylation motif of ALS.

IGFALS on 16p13.3encodes the acid-labile subunit (ALS).ALS forms a ternary complex with insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) and thereby regulates the half-life of IGFs in the circulation 1 .ALS plays a critical role in the growthpromoting effects of growth hormone (GH) 1 .The ALS protein contains seven N-glycosylation motifs with a consensus 3-amino acid sequence (N-X-S/T) [2][3][4] .These motifs mediate the formation of the ternary complex [2][3][4] .To date, more than 60 patients with ALS deficiency resulting from biallelic IGFALS variants have been reported 1 .Most of these patients exhibited moderate short stature due to GH resistance with or without delayed puberty, although several patients achieved low-normal adult height 1,5,6 .Salient endocrine findings of these patients were low levels of IGF-I and IGFBP-3 and normal or elevated levels of GH 1,5 .Known IGFALS variants associated with ALS deficiency include several substitutions and indels widely distributed in the coding region (Fig. 1) 1,[6][7][8][9][10][11][12][13][14][15][16][17][18] .However, only two of these variants (p.S87L and p.N580del) caused changes in the consensus amino acid sequence of the N-glycosylation motifs 7,16 .The pathogenicity of these two variants remains uncertain.This is because the p.S87L variant was coupled with an additional variant in IGF1R, and the p.N580del variant was present only in a heterozygous state 7,16 .In fact, Janosi et al. reported that site-directed mutagenesis to replace the asparagine residue at any of the N-glycosylation motifs with an alanine residue did not alter the in vitro binding activity of ALS to the IGF/IGFBP binary complex 2 .
Here, we report a patient who carried a homozygous 2-amino acid deletion within the fourth N-glycosylation motif of ALS.The patient was a Japanese boy born from a first-cousin couple at 39 weeks and 5 days of gestation.His body weight and length at birth were 2342 g (−2.6 SD) and 47 cm (−1.2 SD), respectively.During childhood, his growth pattern followed the −2.5 SD growth curve of Japanese boys.At 5.2 years of age, he visited our clinic for the evaluation of short stature.Physical examinations showed no phenotypic abnormalities except for short stature (96.6 cm, −2.7 SD).He had no signs of pubertal sexual development.His bone age was 1.5 years younger than his chronological age.Endocrine examinations revealed high basal levels of GH (8.6-22.5 ng/ml; reference range, ≤ 2.5 ng/ml) and a low IGF-I level (17 ng/ml; reference range, 44-193 ng/ml).Arginine stimulation yielded a prominent GH response (82.0 ng/ml; reference range, ≥ 6.0 ng/ml).The levels of thyroid hormones and gonadotropins were within the reference ranges for agematched boys.The heights of his father and mother were 165 cm (−1.0 SDS) and 148 cm (−1.9 SDS), respectively.The height of his 9-year-old sister was −2.0 SDS.
We performed molecular analyses.This study was approved by the Institutional Review Board Committee at the National Center for Child Health and Development.Written informed consent was obtained from the parents of the patient.Genomic DNA samples were obtained from the peripheral leukocytes of the patient and his parents.We performed mutation screening for 16 major growth genes (ACAN, CDKN1C, FGFR3, GH1, GHR, GHRHR, GHSR, GNAS, IGF1, IGF1R, IGF2, IGFALS, JAK2, NPR2, SHOX, and STAT5B) using a nextgeneration sequencer panel (Kazusa DNA Research Institute, Kisarazu, Japan).The methods were described previously 16 .As a result, we identified a homozygous 6-bp deletion (NM_004970.3:c.1103_1108del, p.N368_S370delinsT) in exon 2 of IGFALS.No pathogenic variants were detected in the other genes examined.Sanger sequencing confirmed that the patient had a homozygous IGFALS variant, and his parents were heterozygous carriers (Fig. 2a).The IGFALS variant was not found in the Human Genome Mutation Database (https://www.hgmd.cf.ac.uk/ac/index.php),ClinVar (https:// www.ncbi.nlm.nih.gov/clinvar/), the Genome Aggregation Database (https://gnomad.broadinstitute.org/),or the Japanese Multi Omics Reference Panel (https://jmorp.megabank.tohoku.ac.jp).This variant was classified as a variant of uncertain significance (VUS) based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines (PM2, PM4, PP4) 19 .This variant caused a 3-amino acid deletion and a 1-amino acid insertion in the fourth N-glycosylation motif of ALS (Fig. 2a).Comparative genomic hybridization of the patient using a CGH + SNP array (SurePrint G3 Human Genome Microarray, 2 × 400 k format; Agilent Technologies, Santa Clara, CA, USA) detected loss of heterozygosity (LOH) regions on 10 different chromosomes (Fig. 2b).The total size of the LOH regions was ~187 Mb.The LOH region on chromosome 16 encompassed IGFALS (Fig. 2b).Array-based CGH identified no pathogenic copy number variations.
The boy carried a hitherto unreported variant of IGFALS.The homozygosity of this rare variant can be ascribed to parental consanguinity; the patient carried large LOH regions on 10 chromosomes.The total size of the LOH regions (187 Mb, 6.05% of the entire genome) was similar to the theoretical size of LOH regions in a child born to first-cousin parents (6.25%) 20 .Of note, the heights of the patient (~−2.6 SD) and his heterozygous parents (−1.0 SD and −1.9 SD) were comparable to those of previously reported individuals with biallelic and monoallelic pathogenic variants of IGFALS, respectively (biallelic variants, −2.46 ± 1.07 SD and monoallelic variants, −1.13 ± 1.08 SD) 1 .Moreover, the endocrine data of our patient, such as high GH levels and a low IGF-1 level, were indicative of ALS deficiency 5 .In addition, he had no pathogenic variants in other major growth genes.These results indicate that his phenotype results from the IGFALS variant.The patient may develop delayed  puberty in the future because this is a common manifestation of ALS deficiency 6 .
The IGFALS variant in the patient was a 6-bp deletion.This inframe deletion is unlikely to cause gross conformational changes or mRNA decay; however, this deletion disrupted one of the seven N-glycosylation motifs of ALS.The phenotype of our patient implies that the loss of the fourth N-glycosylation motif abolishes ALS function.Consistent with this, 3D structure prediction suggested that the asparagine residue at the 368 position mediates the binding between ALS and the binary complex 4 .These results highlight the clinical importance of the N-glycosylation motifs of ALS.In conclusion, this study expands the mutation spectrum of IGFALS to include 2-amino acid deletion within an N-glycosylation motif.

HGV DATABASE
The relevant data from this Data Report are hosted at the Human Genome Variation Database at https://doi.org/10.6084/m9.figshare.hgv.3412.

Fig. 1
Fig. 1 Major IGFALS variants identified in patients with short stature.Missense substitutions, indels, and protein-truncating variants are indicated by thin, broken, and thick arrows, respectively.The seven N-glycosylation sites are shown in blue.Variants within N-glycosylation motifs are shown in red.The 2-amino acid deletion in the present case is boldfaced.Brown boxes and the green structure indicate cysteinerich regions and leucine-rich repeats, respectively.

Fig. 2
Fig. 2 Molecular data of the patient.a Sanger sequencing showing the c.1103_1108del, p.N368_S370delinsT variant.The parents of the patient were heterozygous carriers.The 6-bp deletion was located within the fourth N-glycosylation motif (N-L-S).b The results of comparative genomic hybridization using a SNP + CGH array.The gray-shaded areas depict regions of loss of heterozygosity (LOH).The LOH region on chromosome 16 included IGFALS.